Tight junctions (TJs) are essential cellular structures that form the selective paracellular barriers in endothelial and epithelial cells, including the blood-brain, and blood-retinal barriers, lung and gut epithelium. The barrier properties of TJs regulate metabolite flux, fluid flow and drug uptake into these tissues. Dysfunction in TJ barrier properties is observed in a large number of human diseases including cancer (e.g. brain tumors), stroke, retinopathies, kidney disorders, irritable bowel syndrome, Alzheimer's disease and vascular dementia, and asthma. Moreover, TJ components are used as cellular receptors by a range of pathogens. Thus, an improved understanding of the basic structural and functional biology of TJs will have wide ranging impact for human health. Specifically, this information may lead to improved drug delivery methods, novel protocols for regulating TJ permeability in disease and prevention of some infections. Currently, little is known about the molecular basis for TJ barrier properties and how they are regulated. In this proposal, we describe a combined structural, biochemical and cellular and molecular approach to illuminate the function of occludin (Occ), a transmembrane component of TJs, which has been implicated in the regulation of TJ barrier properties. These studies build upon our previously published and unpublished data implicating Occ, and its binding partner zona occludens 1 (ZO-1) protein, in regulating barrier properties conferred by TJs. The planned studies are organized into three specific aims. In Aim1, experiments are described, using a cell biological approach to determine the molecular mechanism(s) for the effects of phosphorylation of Serine 471 in Occ (S471) on TJ cellular structure function. In Aim2, we describe experiments to determine the structural details of the ZO-1/Occ protein-binding core, which includes S471, and with their functional binding complexes In Aim 3, we extend these approaches to determine the contribution of S471 phosphorylation of Occ to its interaction with ZO-1 using full length Occ and dimeric ZO-1 constructs. Together, the results of these three aims will provide a structural and biochemical basis for understanding Occ function and starting point for developing novel strategies for modulating TJ barrier properties that target the ZO-1/Occ complex.